<p>A/ The Biacore experiment was performed on a CM5 chip sensitized by scFv 2LF. The curve ... more <p>A/ The Biacore experiment was performed on a CM5 chip sensitized by scFv 2LF. The curve corresponding to the LF variant in which LF(97–103) was shaved is in purple, the curves corresponding to the shaving of LF(136–143), LF(178–184), LF(227–231) and LF(231–236) are in green, blue, red and orange, respectively. The positive control (commercially available LF) curve is in gray. Only the reactivities of LF variants in which LF(178–184), LF(227–231), LF(231–236) had been shaved were significantly (three-fold or more) lower than the positive control value. B/ The same color code as in A indicated the five epitope candidates on an apical view of the LF<sub>N</sub> surface. The three regions selected after alanine shaving, LF(178–184), LF(227–231) and LF(231–236), are proximal. The figure was drawn using Swiss PDB viewer and Pov-ray rendering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone.0065855-Kaplan1" target="_blank">[49]</a>.</p
Toxins necessary for Bacillus anthracis pathogenesis are made of three sub-units : PA (protective... more Toxins necessary for Bacillus anthracis pathogenesis are made of three sub-units : PA (protective antigen), LF (lethal factor) and EF (edema factor). Anti-PA recombinant antibodies have been developed for anthrax treatment, but other sub-units have not been targeted despite anthrax experts recommendations. Here, we describe an anti-LF scFv that was obtained from a macaque (Macaca fascicularis) immune antibody gene library (1.8x10 8 clones) in pHAL14 phagemid vector. One scFv clone (2LF) selected from the library, had a high-affinity (KD = 1.02 nM), was highly neutralizing in the standardized in vitro (IC50 = 1.17 ± 0.06 nM) and in an in vivo assays. The genes encoding 2LF are similar to human immunoglobulin germline genes, and assigned to subgroups of human V, (D) or J genes by IMGT/V-QUEST. 2LF framework regions have a 84% identity with their most similar, germinally encoded human counterparts. This scFv neutralizes the anthrax lethal toxin by inhibiting the formation of the LF-PA complex, as shown in a competition assay. This inhibition suggests that 2LF interacts with domain 1 of LF, which is partially shared with EF and 2LF also reacted with EF, in ELISA and SPR. A 2LF-derived IgG, targeting LF and maybe EF, would be suitable for medical use.
<p>A CM5 chip was sensitized with scFv 2LF. LF (control, in grey) and its five variants in ... more <p>A CM5 chip was sensitized with scFv 2LF. LF (control, in grey) and its five variants in which H229 (yellow), R230 (blue), Q234 (red), L235 (green) or Y236 (brown) had been individually mutated in alanine reacted in flux. The LF variant in which these five positions had been simultaneously mutated in alanine is presented in blue. Dilutions of LF and its variants giving an equivalent maximal signal are presented, to allow the comparison of the dissociation phases (after the maximal signal and the artefactual spikes) when dissociation constants are measured (between the 500<sup> th</sup> and 700<sup>th</sup> seconds). Values were calculated on several curves for each variant, and are presented on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone-0065855-t002" target="_blank">table 2:</a> the five variants with punctual mutations present at least a three-fold more rapid dissociation than the control, and the variant with the five simultaneous mutations presents no reactivity.</p
<p>The α carbons constituting LF are drawn as a gray tube, and PA is represented as its mol... more <p>The α carbons constituting LF are drawn as a gray tube, and PA is represented as its molecular surface (pink). The region LF(178–184), the shaving of which abolished reactivity between scFv 2LF and LF, is represented by a yellow tube. Residues of the core of the LF epitope are shown as sticks, colored according to the atom type (carbon in cyan), and are located on α helix 10. The figure was drawn using VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone.0065855-Humphrey1" target="_blank">[50]</a>.</p
Anthrax is an acute disease caused by the bacterium Bacillus anthracis, and is a potential biowar... more Anthrax is an acute disease caused by the bacterium Bacillus anthracis, and is a potential biowarfare/bioterrorist agent. Its pulmonary form, caused by inhalation of the spores, is highly lethal and is mainly related to injury caused by the toxins secretion. Antibodies neutralizing the toxins of B. anthracis are regarded as promising therapeutic drugs, and two are already approved by the Federal Drug Administration. We developed a recombinant human-like humanized antibody, 35PA83 6.20, that binds the protective antigen and that neutralized anthrax toxins in-vivo in White New Zealand rabbits infected with the lethal 9602 strain by intranasal route. Considering these promising results, the preclinical and clinical phase one development was funded and a program was started. Unfortunately, after 5 years, the preclinical development was cancelled due to industrial and scientific issues. This shutdown underlined the difficulty particularly, but not only, for an academic laboratory to proc...
This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit:
Background: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently ... more Background: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. Results: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies ...
Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by sing... more Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phagedisplayed library of macaque origin
Rapidly after the clinical success of the first murine therapeutic antibody licensed in 1985 (mur... more Rapidly after the clinical success of the first murine therapeutic antibody licensed in 1985 (muromomab-CD3), the first limits of the therapeutic use of antibodies deriving from hybridoma technology appeared. Indeed, the nonhuman nature of these therapeutic antibodies makes them immunogenic when administrated to patients, which develop anti-drug antibodies (ADA). If repeated drug-administrations are needed, the immune response will accelerate the elimination of the drug, leading to a therapeutic failure, or in the worst case to an anaphylactic reaction against the murine protein. Several antibody generations were then developed to obtain better-tolerated molecules: chimeric, humanized, and fully human antibodies. The first antibody generation is fully based on cellular technology (mice hybridoma technology), but the next generations are improved by molecular engineering. Immune antibody phage-display libraries are one successful approach to isolating such engineered antibodies. One ...
Antibodies (Abs) may significantly improve the outcome of diseases caused by toxins of military i... more Antibodies (Abs) may significantly improve the outcome of diseases caused by toxins of military interest, in particular ricin and botulism toxins. The efficacy of Abs to neutralize ricin was demonstrated in vivo utilizing Abs of animal origin but recombinant antibodies (rAbs), which would be better tolerated, are preferred for clinical use. Animal Abs are utilized at present as medical countermeasure to neutralize botulism toxins, but they show limitations and rAbs are also preferred for this clinical use. Of note, anthrax is an infective disease depending on toxins for its pathogenicity, and it was demonstrated that anti-anthrax toxins Abs could be of benefit when used as adjunct to antibiotics. We used an original strategy, starting from libraries derived from lymphocytes of non-human primates (NHP) immunized with the toxin of interest, to isolate Abs directed against all these toxins. The libraries are screened by the phage-display technology to isolate the best candidates, which are then tested for their toxin neutralization properties. Abs neutralizing the anthrax lethal toxin (35PA83, 2LF) and ricin (43RCA) have been isolated and at present, Abs neutralizing the botulinum toxins (BoNT) A, B and E are being isolated.
Fab 35PA 83 is an antibody fragment of non-human primate origin that neutralizes the anthrax leth... more Fab 35PA 83 is an antibody fragment of non-human primate origin that neutralizes the anthrax lethal toxin. Human antibodies are usually preferred when clinical use is envisioned, even though their framework regions (FR) may carry mutations introduced during affinity maturation. These hypermutations can be immunogenic and therefore FR that are encoded by human germline genes, encountered in IgMs and thus part of the "self" proteins, are preferable. Accordingly, the proportion of FR residues in 35PA 83 that were encoded by human V and J germline genes, i.e. the germinality index (GI) of 35PA 83 , was increased in a multistep cumulative approach. In a first step, the FR1 and FR4 residues of 35PA 83 were changed simultaneously into their counterparts coded by 35PA 83 's closest human germline genes, without prior modelling. The resulting derivative of 35PA 83 had the same affinity as its parental Fab. In a second step, the 3D structures of this first 35PA 83 derivative, carrying the same type of residue changes but in the FR2 and FR3 regions, were modelled in silico from sequences. Some of the changes in FR2 or FR3 modified the predicted peptide backbone. The changes that did not seem to alter the structure were introduced simultaneously in the Fab by an in vitro method and resulted in a loss of reactivity, which could however be fully restored by a single point mutation. The final 35PA 83 derivative had a GI higher than that of a fully human Fab, which had neutralization properties similar to 35PA 83 and which was used as a benchmark in this study.
Pseudomonas aeruginosa is a gram-negative pathogen causing life-threatening infections. Lung inju... more Pseudomonas aeruginosa is a gram-negative pathogen causing life-threatening infections. Lung injury and the development of sepsis depend largely on the expression of type III secretion system (TTSS) virulence. TTSS functions as a molecular syringe to deliver toxins directly to the cytosol of cells, inhibit innate immune mechanisms, and prevent bacterial clearance. Polyclonal antibodies that bind to PcrV of P. aeruginosa inhibit the delivery of type III toxins and enhance the clearance of bacteria during acute lung infections. PcrV is a homologue of LcrV, a protective antigen in the Yersinia TTSS and an integral component of TTSS. In this study, a murine monoclonal antibody (MAb) to PcrV was generated: MAb 166, which is protective against P. aeruginosa when coinstilled with the bacterial inoculum or intraperitoneally transferred to mice. Fab fragments from MAb 166 prevent sepsis and death. The epitope bound by MAb 166 was mapped to the carboxyl-terminus of PcrV.
Background Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic s... more Background Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use. Results Diversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screeni...
<p>A/ The Biacore experiment was performed on a CM5 chip sensitized by scFv 2LF. The curve ... more <p>A/ The Biacore experiment was performed on a CM5 chip sensitized by scFv 2LF. The curve corresponding to the LF variant in which LF(97–103) was shaved is in purple, the curves corresponding to the shaving of LF(136–143), LF(178–184), LF(227–231) and LF(231–236) are in green, blue, red and orange, respectively. The positive control (commercially available LF) curve is in gray. Only the reactivities of LF variants in which LF(178–184), LF(227–231), LF(231–236) had been shaved were significantly (three-fold or more) lower than the positive control value. B/ The same color code as in A indicated the five epitope candidates on an apical view of the LF<sub>N</sub> surface. The three regions selected after alanine shaving, LF(178–184), LF(227–231) and LF(231–236), are proximal. The figure was drawn using Swiss PDB viewer and Pov-ray rendering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone.0065855-Kaplan1" target="_blank">[49]</a>.</p
Toxins necessary for Bacillus anthracis pathogenesis are made of three sub-units : PA (protective... more Toxins necessary for Bacillus anthracis pathogenesis are made of three sub-units : PA (protective antigen), LF (lethal factor) and EF (edema factor). Anti-PA recombinant antibodies have been developed for anthrax treatment, but other sub-units have not been targeted despite anthrax experts recommendations. Here, we describe an anti-LF scFv that was obtained from a macaque (Macaca fascicularis) immune antibody gene library (1.8x10 8 clones) in pHAL14 phagemid vector. One scFv clone (2LF) selected from the library, had a high-affinity (KD = 1.02 nM), was highly neutralizing in the standardized in vitro (IC50 = 1.17 ± 0.06 nM) and in an in vivo assays. The genes encoding 2LF are similar to human immunoglobulin germline genes, and assigned to subgroups of human V, (D) or J genes by IMGT/V-QUEST. 2LF framework regions have a 84% identity with their most similar, germinally encoded human counterparts. This scFv neutralizes the anthrax lethal toxin by inhibiting the formation of the LF-PA complex, as shown in a competition assay. This inhibition suggests that 2LF interacts with domain 1 of LF, which is partially shared with EF and 2LF also reacted with EF, in ELISA and SPR. A 2LF-derived IgG, targeting LF and maybe EF, would be suitable for medical use.
<p>A CM5 chip was sensitized with scFv 2LF. LF (control, in grey) and its five variants in ... more <p>A CM5 chip was sensitized with scFv 2LF. LF (control, in grey) and its five variants in which H229 (yellow), R230 (blue), Q234 (red), L235 (green) or Y236 (brown) had been individually mutated in alanine reacted in flux. The LF variant in which these five positions had been simultaneously mutated in alanine is presented in blue. Dilutions of LF and its variants giving an equivalent maximal signal are presented, to allow the comparison of the dissociation phases (after the maximal signal and the artefactual spikes) when dissociation constants are measured (between the 500<sup> th</sup> and 700<sup>th</sup> seconds). Values were calculated on several curves for each variant, and are presented on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone-0065855-t002" target="_blank">table 2:</a> the five variants with punctual mutations present at least a three-fold more rapid dissociation than the control, and the variant with the five simultaneous mutations presents no reactivity.</p
<p>The α carbons constituting LF are drawn as a gray tube, and PA is represented as its mol... more <p>The α carbons constituting LF are drawn as a gray tube, and PA is represented as its molecular surface (pink). The region LF(178–184), the shaving of which abolished reactivity between scFv 2LF and LF, is represented by a yellow tube. Residues of the core of the LF epitope are shown as sticks, colored according to the atom type (carbon in cyan), and are located on α helix 10. The figure was drawn using VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065855#pone.0065855-Humphrey1" target="_blank">[50]</a>.</p
Anthrax is an acute disease caused by the bacterium Bacillus anthracis, and is a potential biowar... more Anthrax is an acute disease caused by the bacterium Bacillus anthracis, and is a potential biowarfare/bioterrorist agent. Its pulmonary form, caused by inhalation of the spores, is highly lethal and is mainly related to injury caused by the toxins secretion. Antibodies neutralizing the toxins of B. anthracis are regarded as promising therapeutic drugs, and two are already approved by the Federal Drug Administration. We developed a recombinant human-like humanized antibody, 35PA83 6.20, that binds the protective antigen and that neutralized anthrax toxins in-vivo in White New Zealand rabbits infected with the lethal 9602 strain by intranasal route. Considering these promising results, the preclinical and clinical phase one development was funded and a program was started. Unfortunately, after 5 years, the preclinical development was cancelled due to industrial and scientific issues. This shutdown underlined the difficulty particularly, but not only, for an academic laboratory to proc...
This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit:
Background: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently ... more Background: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. Results: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies ...
Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by sing... more Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phagedisplayed library of macaque origin
Rapidly after the clinical success of the first murine therapeutic antibody licensed in 1985 (mur... more Rapidly after the clinical success of the first murine therapeutic antibody licensed in 1985 (muromomab-CD3), the first limits of the therapeutic use of antibodies deriving from hybridoma technology appeared. Indeed, the nonhuman nature of these therapeutic antibodies makes them immunogenic when administrated to patients, which develop anti-drug antibodies (ADA). If repeated drug-administrations are needed, the immune response will accelerate the elimination of the drug, leading to a therapeutic failure, or in the worst case to an anaphylactic reaction against the murine protein. Several antibody generations were then developed to obtain better-tolerated molecules: chimeric, humanized, and fully human antibodies. The first antibody generation is fully based on cellular technology (mice hybridoma technology), but the next generations are improved by molecular engineering. Immune antibody phage-display libraries are one successful approach to isolating such engineered antibodies. One ...
Antibodies (Abs) may significantly improve the outcome of diseases caused by toxins of military i... more Antibodies (Abs) may significantly improve the outcome of diseases caused by toxins of military interest, in particular ricin and botulism toxins. The efficacy of Abs to neutralize ricin was demonstrated in vivo utilizing Abs of animal origin but recombinant antibodies (rAbs), which would be better tolerated, are preferred for clinical use. Animal Abs are utilized at present as medical countermeasure to neutralize botulism toxins, but they show limitations and rAbs are also preferred for this clinical use. Of note, anthrax is an infective disease depending on toxins for its pathogenicity, and it was demonstrated that anti-anthrax toxins Abs could be of benefit when used as adjunct to antibiotics. We used an original strategy, starting from libraries derived from lymphocytes of non-human primates (NHP) immunized with the toxin of interest, to isolate Abs directed against all these toxins. The libraries are screened by the phage-display technology to isolate the best candidates, which are then tested for their toxin neutralization properties. Abs neutralizing the anthrax lethal toxin (35PA83, 2LF) and ricin (43RCA) have been isolated and at present, Abs neutralizing the botulinum toxins (BoNT) A, B and E are being isolated.
Fab 35PA 83 is an antibody fragment of non-human primate origin that neutralizes the anthrax leth... more Fab 35PA 83 is an antibody fragment of non-human primate origin that neutralizes the anthrax lethal toxin. Human antibodies are usually preferred when clinical use is envisioned, even though their framework regions (FR) may carry mutations introduced during affinity maturation. These hypermutations can be immunogenic and therefore FR that are encoded by human germline genes, encountered in IgMs and thus part of the "self" proteins, are preferable. Accordingly, the proportion of FR residues in 35PA 83 that were encoded by human V and J germline genes, i.e. the germinality index (GI) of 35PA 83 , was increased in a multistep cumulative approach. In a first step, the FR1 and FR4 residues of 35PA 83 were changed simultaneously into their counterparts coded by 35PA 83 's closest human germline genes, without prior modelling. The resulting derivative of 35PA 83 had the same affinity as its parental Fab. In a second step, the 3D structures of this first 35PA 83 derivative, carrying the same type of residue changes but in the FR2 and FR3 regions, were modelled in silico from sequences. Some of the changes in FR2 or FR3 modified the predicted peptide backbone. The changes that did not seem to alter the structure were introduced simultaneously in the Fab by an in vitro method and resulted in a loss of reactivity, which could however be fully restored by a single point mutation. The final 35PA 83 derivative had a GI higher than that of a fully human Fab, which had neutralization properties similar to 35PA 83 and which was used as a benchmark in this study.
Pseudomonas aeruginosa is a gram-negative pathogen causing life-threatening infections. Lung inju... more Pseudomonas aeruginosa is a gram-negative pathogen causing life-threatening infections. Lung injury and the development of sepsis depend largely on the expression of type III secretion system (TTSS) virulence. TTSS functions as a molecular syringe to deliver toxins directly to the cytosol of cells, inhibit innate immune mechanisms, and prevent bacterial clearance. Polyclonal antibodies that bind to PcrV of P. aeruginosa inhibit the delivery of type III toxins and enhance the clearance of bacteria during acute lung infections. PcrV is a homologue of LcrV, a protective antigen in the Yersinia TTSS and an integral component of TTSS. In this study, a murine monoclonal antibody (MAb) to PcrV was generated: MAb 166, which is protective against P. aeruginosa when coinstilled with the bacterial inoculum or intraperitoneally transferred to mice. Fab fragments from MAb 166 prevent sepsis and death. The epitope bound by MAb 166 was mapped to the carboxyl-terminus of PcrV.
Background Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic s... more Background Botulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use. Results Diversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screeni...
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